1. Field of the Invention
The present invention provides methods for treating or preventing vascular proliferative diseases in vivo by administration of a gene which encodes p27.
2. Discussion of the Background
Vascular diseases are characterized by a fibroproliferative response to injury consisting of smooth muscle cell proliferation and migration as well as connective tissue formation. The mechanisms by which vascular smooth muscle cells (VSMC) proliferate in response to mitogenic signals are well described; however, the role of cellular gene products which cause VSMC to shift from a proliferative to a nonproliferative state during G1 phase of the cell cycle are not well understood.
It is known that transitions between phases of the cell cycle are catalyzed by a family of cyclin-dependent kinases (P. Nurs, (1990) Nature 344, 503-508; L. Hartwell et al., (1974) Science 183 46-51). In many cells, transit through G1 of the cell cycle and entry into S phase requires a binding and activation of cyclin/cyclin-dependent kinase complexes (CDK), predominantly cyclin D-cdk4,6 and cyclin E-cdk2 (C. J. Sherr, (1994) Cell 79, 551; C. J. Sherr (1996) Science 274, 1672).
The cyclin-dependent kinase inhibitors (CKIs) are naturally-occurring gene products which inhibits cyclin-CDK activity and phosphorylation of retinoblastoma (Rb), resulting in G1/S growth arrest (D. O. Morgan, Nature 1995, 374: 171; C. J. Sherr & J. M. Roberts, Genes. Dev. 1995, 9: 1149). CKIs directly implicated in CDK regulation are p21cipI/WafI (Y. Xiong, et al., Nature 1993, 366: 701; J. W. Harper et al., Cell 1993, 75: 805), p27KipI (H. Pyoshima, T. Hunter, Cell 1994, 78: 67; K. Polyak et al., Cell 1994, 78: 59; S. Coats et al., Science 1996, 272; 877), and p16/p15INKN (M. Serrano et al., Nature 1993, 366: 704).
Previous studies of these CKIs were focused on their potential role in malignant transformation. For example, PCT Publication No. WO95/18824 (applicant Sloan-Kettering Institute For Cancer Research) describes a method for identifying agents capable of modulating the ability of p27 to inhibit the activation of the cyclin E-Cdk2 complex. This PCT publication further provides methods for treating subjects diagnosed with a hyperproliferative disorder, such as cancer and hyperplasia, using these agents. Such agents can be both protein and non-protein moieties. Unfortunately, the involvement of CKIs in cardiovascular diseases, including atherosclerosis, angiogenesis and restenosis, has not been well studied.
There are currently a number of methods used to treat cardiovascular diseases which focus on inhibiting cell proliferation. The main problems associated with the available therapies revolve around targeting the inhibitory agent to the proliferating cells that need to be killed. Targeting has traditionally been attempted using chemo- or radiotherapeutic agents coupled with antibodies. More recently, gene therapy approaches have been used that target proliferating cells by providing gene products detrimental to specific cell types. The genes encoding these gene products are known as suicide genes. The gene products are either instilled site specifically, expressed in specific cells using vectors which target specific cells or expressed under the control of a cell type-specific promoter.
The HSV-1 thymidine kinase gene (TK) is the most widely used suicide gene in mammalian systems. TK efficiently phosphorylates guanosine analogs ganciclovir (GCV) and acyclovir (ACV), which are subsequently phosphorylated by cellular enzymes into their triphosphate forms. These end-products are incorporated into the growing DNA chain, leading to elongation arrest (ACV) or a drastic slow down in DNA synthesis (GCV). Death usually ensues, through a mechanism identified in some cell lines as apoptosis. The mechanism that triggers cell death is not known. In the case of GCV, another action other than at the level of the DNA polymerase inhibition might exist, since no correlation is observed between the inhibition of mutant viral DNA polymerase by GCV and growth of these mutants in the presence of GCV.
One of the peculiar features of the TK-GCV system is the bystander effect that characterized the death of untransduced cells. Two mechanisms have been proposed to explain this phenomenon. In 1993, Freeman and colleagues hypothesized that the uptake of phosphorylated GCV by bystander cells occurs via the endocytosis of apoptotic vesicles, originating from the TK-transduced cells, and containing the toxic drug. However increasing evidence suggests that the bystander effect is mediated via gap junctional intercellular communications, that allow phosphorylated ganciclovir to translocate from TK+ to TK− cells. In 1995, using a flow cytometry assay to quantitate cellar coupling, Fick and colleagues found that bystander tumor cytotoxicity during GCV treatment was highly correlated with the exent of gap junction-mediated coupling. In a TK-expressing neuro-2a murine neuroblastoma cell line, which do not normally exhibit any bystander effect, adenovirus-mediated overexpression of connexin-43 was shown to confer bystander effect-mediated cell killing.
The TK-GCV system has already been successfully applied in cancer models as well as restenosis in vivo (Plautz et al., Circulation 1991, 83:578; Ohno et al., Science 1994, 265:781). However, the efficiency of gene delivery in vivo remains very low, and enhancement of TK-mediated killing at other levels must be considered. In an attempt to link TK-mediated tumor suppression and immune system, retroviral vectors were constructed carrying both the HSV-TK and interleukin-2 genes. However, using a rat 9L gliosarcoma model, no enhancement of tumor eradication was observed with this vector.